Antigens Derived from Citrullinated 14-3-3 and Uses Thereof in the Diagnosis of Rheumatoid Arthritis

ABSTRACT

The present invention provides citrullinated 14-3-3η peptides and antibodies thereto and methods of using same to evaluate arthritic conditions such as rheumatoid arthritis.

FIELD OF INVENTION

The present invention relates generally to citrullinated peptidesspecifically bound by serum autoantibody, and anti-14-3-3 autoantibodiesin particular, present in the serum of patients suffering from arthriticconditions. Such peptides comprise a citrullinated 14-3-3 eta sequence,or portion thereof, in which an arginine in the native sequence has beendeiminated to a citrulline. The invention also concerns the use of thesepeptides to evaluate arthritic conditions including rheumatoid arthritisand antibodies directed to these citrullinated peptides.

BACKGROUND OF THE INVENTION

Arthritis, or arthralgia, generally refers to inflammatory disorders ofthe joints of the body, and is usually accompanied by pain, swelling andstiffness. Arthritis may result from any of several causes includinginfections, trauma, degenerative disorders, metabolic disorders ordisturbances or other unknown etiologies.

Rheumatoid Arthritis (RA), for example, is a chronic inflammatorydisorder of the synovial membranes, and is one of the most commonsystemic autoimmune diseases. The diagnosis of RA depends primarily onclinical manifestations, but laboratory results are helpful indifferential diagnosis and disease management. Early diagnosis of RA isimportant both in disease treatment and management. Kim and Weisman(2000) Arthritis. Rheum. 43:473-84.

The serum of affected patients contains factors that may be markers forthe disease, allowing for early diagnosis. Historically, rheumatoidfactor (RF) has long been one of the primary serologic indicators forRA. Additionally, anti-keratin autoantibodies (AKA), also known asanti-perinuclear autoantibodies, are detected in 40-55% of RA patientsand in 40-50% of clinically diagnosed RA patients who are RF negative.Vincent et al. (1989) Ann. Rheum. Dis. 48:712-22; Corconnier et al.(1996) Br. J. Rheumatol. 35:620-4; Gabay et al. (1993) Ann. Rheum. Dis.52:785-9. AKA is generally considered to be significantly more specificthan RF. Additionally, AKA may precede the clinical appearance of RA bymonths or years. PCT Publication No. 2010102412 entitled “Compositionsand Methods for Characterizing Arthritic Conditions” also describesautoantibodies to 14-3-3 proteins and methods of using them to evaluatearthritic conditions such as RA. For example, 14-3-3 eta is normally anintracellular protein and only in the disease state is it released intothe extracellular space. As such, serum 14-3-3 eta and/or autoantibodiesto same have diagnostic utility as markers that complement otherserologic indicators in early and established RA and are associated withjoint damage in RA and PsA,

Recently it was determined that AKA recognize an epitope that containscitrulline. von Venrooj (2000) Arthritis Res. 52:785-9. Citrullinationis a form of a post-translational modification (PTM) wherebypeptidylarginine deiminases (PAD) catalyze the deimination of the aminoacid arginine (R) to citrulline (C) resulting in a chemical changeliberating a nitrogen-based moiety. Disregulated citrullination appearsto be an active process in inflammatory conditions like RA whereby an“insult” results in 1) activation of PAD; 2) release of PAD enzymes intothe synovial space; 3) citrullination of extracellular proteins likevimentin and filaggrin; 4) a humoral immune response against thecitrullinated antigens and 5) the perpetuation of the disease. Detectionof these anti-citrullinated antibodies may also be useful in the earlydiagnosis of RA.

IgG antibodies against a synthetic peptide containing citrulline knownas CCP (Cyclic Citrullinated Peptide) has proven to be better thaneither AKA or RF testing in differentiating RA from other autoimmunediseases, and the presence of anti-CCP antibody occurs independently ofelevated RF levels in patients with RA. However, a significantpercentage of patients are or remain seronegative for anti-CCP.Accordingly, there remains a significant need in the art for better andmore specific diagnostic indicators of this disease.

SUMMARY OF INVENTION

As disclosed herein, the citrullination sites of human 14-3-3 eta wereidentified in silico, in vitro, and using clinical samples. Thecitrullinated form of 14-3-3 eta and/or anti-14-3-3 autoantibodiesspecific for these post-translational modifications compared to thenative or non-citrullinated form of 14-3-3 eta may be used for thediagnosis and prognosis of rheumatoid arthritis, including in anti-CCPnegative patients. In particular, and as disclosed herein for the firsttime, 14-3-3 eta represents a novel citrullination target that isdifferentially expressed in anti-CCP negative RA patients versus healthycontrols, indicating that detection of the citrullinated protein, and/orautoantibodies specific for citrullinated-14-3-3 eta may significantlyimprove RA diagnosis.

Accordingly, the invention provides compositions comprising acitrullinated 14-3-3 eta protein or a citrullinated fragment thereof andmethods of using same, e.g., for the diagnosis and prognosis ofrheumatoid arthritis. Also provided herein are compositions comprisingmonoclonal antibodies that selectively bind to a citrullinated 14-3-3eta protein and/or to a specific citrullination site within saidprotein, as opposed to the corresponding native or noncitrullinatedform, and methods of using same.

In one aspect, the present invention concerns the finding that thepresence/quantity of autoantibodies directed against the citrullinatedform of 14-3-3 eta in a biological sample is indicative of the existenceand/or status of the arthritic condition in the subject. Also providedherein are methods for evaluating and/or characterizing an arthriticcondition in a mammalian subject comprising detecting circulating immunecomplexes with at least one citrullinated 14-3-3 eta protein or afragment thereof in a biological sample from a subject.

In preferred embodiments, the citrullinated 14-3-3 eta protein orfragment thereof comprises at least one citrulline residue at at leastone citrullination site, preferably selected from the group consistingof position 4, position 12, position 19, position 42, position 61,position 86 or position 227 of the native 14-3-3 eta sequence, orcombinations thereof. The citrullinated 14-3-3 eta protein or fragmentthereof may be isolated or, more preferably, bound to a solid support asdescribed in more detail herein.

Accordingly, described herein are methods for evaluating and/orcharacterizing an arthritic condition in a mammalian subject comprisingcontacting a biological sample from the subject with at least onecitrullinated 14-3-3 eta protein or fragment thereof and detecting anautoantibody against the citrullinated 14-3-3 eta protein or fragmentthereof, wherein the presence/quantity of an autoantibody against saidat least one citrullinated 14-3-3 eta protein or fragment thereof isindicative of the existence and/or status of the arthritic condition inthe subject. Also provided herein are methods for evaluating and/orcharacterizing an arthritic condition in a mammalian subject comprisingdetecting circulating immune complexes between an autoantibody and atleast one citrullinated 14-3-3 eta protein in a biological sample fromthe subject, wherein the presence/quantity of existing immune complexesin the sample is indicative of the existence and/or status of thearthritic condition in the subject.

In one embodiment, the detecting step includes quantifying/measuring thelevel of autoantibodies against, or immune complexes with, citrullinated14-3-3 eta protein or a fragment thereof in the biological sample forcomparison with a control sample. Accordingly, the presently-claimedmethods for evaluating an arthritic condition in a subject may provideprognostic as well as diagnostic determinations.

In one aspect, the control sample is a normal control, and thecomparison is indicative of an arthritis diagnosis. In one embodiment,an increased level of autoantibody against, or immune complexes with,citrullinated 14-3-3 eta protein or a fragment thereof in saidbiological sample in comparison with a normal control sample (e.g., fromanother subject not having an arthritic condition) is a diagnosticindicator of an arthritic condition in said subject.

Accordingly, in some embodiments, the presence of autoantibodies tocitrullinated 14-3-3 eta protein or immune complexes thereof in thebiological sample from the subject and/or the presence of an increasedlevel of such autoantibodies or immune complexes in the biologicalsample from the subject relative to a level of such autoantibodies orimmune complexes in a normal (i.e. non-arthritic) control sampleprovides a diagnosis that the subject has an arthritic condition.

In one aspect, the control sample is a previous biological sample fromthe mammalian subject, and the comparison is indicative of diseaseprogression and/or efficacy of a therapeutic regimen. In one embodiment,a decreased level of autoantibodies to citrullinated 14-3-3 eta orcirculating immune complexes thereof in said sample compared to theprevious sample (e.g., a baseline biological sample from said subject)is indicative of the efficacy of an ongoing therapeutic regimen. Inanother embodiment, an increased level of autoantibodies tocitrullinated 14-3-3 eta or circulating immune complexes thereof in saidsample compared to the previous sample is indicative of a lack of aresponse to a therapeutic regimen.

Accordingly, in some embodiments, the relative level of autoantibodiesagainst, or immune complexes with, citrullinated 14-3-3 eta or afragment thereof detected in the biological sample from the subjectcompared to the level of such autoantibodies or complexes present in abaseline biological sample from the same subject provides a prognosticindication of the arthritic condition, and/or a theranostic indicationof the potential efficacy of a proposed therapeutic regimen.

In one aspect, the control sample is the same biological sample from themammalian subject, and the comparison is with the relative level oramount of autoantibodies directed to native or non-citrullinated 14-3-3eta, which can also be indicative of disease progression and/or theefficacy of a potential therapeutic regimen as described herein. In oneembodiment, increased levels of autoantibodies to citrullinated 14-3-3eta or circulating immune complexes thereof in said sample compared tothe levels of autoantibodies to native or non-citrullinated 14-3-3 etaor circulating immune complexes thereof in the same sample is indicativeof the staging and/or prognosis of the disease, or suggestive ofpotential therapeutic interventions (e.g. inhibitors that directlytarget peptidyl arginine deminiases and the like).

Accordingly, in some embodiments, the relative level of autoantibodiesagainst, or immune complexes with, citrullinated 14-3-3 eta or afragment thereof detected in the biological sample from the subjectcompared to the level of autoantibodies or complexes to the native ornon-citrullinated form in the same or sequential biological sample(s)from the subject provides a prognostic indication of the arthriticcondition, and/or a theranostic indication of the potential efficacy ofa proposed therapeutic regimen.

In one aspect, the control sample is an arthritic control, and thecomparison is indicative of disease prognosis. In one embodiment, therelative level of autoantibodies to citrullinated 14-3-3 eta or immunecomplexes thereof in comparison to an arthritic control sample (e.g.,from another subject with a well-defined arthritic condition) is aprognostic indicator of arthritis.

Accordingly, in some embodiments, subjects with different arthriticstatus have detectable differences in levels of autoantibodies to atleast one citrullinated 14-3-3 eta protein or fragment thereof, and/orcirculating immune complexes of such, and these differences are ofprognostic relevance. In one example, disclosed herein are methods thatmay be used to determine a specific disease stage or thehistopathological phenotype of an arthritic condition based on therelative level of autoantibody detected in a subject compared to levelspreviously determined to exist throughout the course of the arthriticcondition, e.g., before treatment, during treatment, after treatment, inanother patient, etc. In another example, the methods disclosed hereinmay be used to classify a biological sample as being from a subject athigh risk for manifestation of an arthritic condition based on therelative level of autoantibodies detected in the biological samplecompared to a control sample, which may be, e.g., stored in a database.

In another aspect, the methods disclosed herein may be used fortheranostic purposes, e.g., to predict the responsiveness of a subjectto a proposed therapeutic regimen based on the relative level ofautoantibodies detected in a biological sample from the subject comparedto a control sample, e.g., of a second biological sample from a secondsubject that was successfully treated with the proposed therapeuticregimen.

Accordingly, in some embodiments, the relative level of autoantibodiesagainst, or immune complexes with, at least one citrullinated 14-3-3 etaprotein or fragment thereof in the biological sample from the firstsubject is compared to the level of autoantibodies against, or immunecomplexes with, citrullinated 14-3-3 eta in biological samples fromsubjects whose abilities to respond to a treatment are known, whereinsuch comparison determines the response potential of the first subjectto the treatment. Determination of the sensitivity of the subject to atherapeutic regimen may then be used to inform methods of treating asubject with an arthritic condition. For example, described herein aremethods of treating a subject with an arthritic condition comprisingmeasuring the level of autoantibody against citrullinated 14-3-3 eta ina biological sample from the subject (e.g., by measuring the level ofautoantibody/citrullinated 14-3-3 eta immune complex formation),correlating the level of autoantibody against or immune complex withcitrullinated 14-3-3 eta with sensitivity of the subject to atherapeutic regimen, and providing the therapeutic regimen to thesubject. In one aspect, the invention provides methods for monitoringtreatment of an arthritic condition, comprising determining the level ofautoantibodies against, or immune complexes with, at least onecitrullinated 14-3-3 eta protein or fragment thereof in patient samplesand monitoring the level of autoantibodies/immune complexes involvingcitrullinated 14-3-3 eta in a patient undergoing treatment.

In another aspect, provided herein are methods for determining and/ordifferentiating the subtypes of arthritis in a patient. In this aspect,the relative level of autoantibodies against, or immune complexes with,at least one citrullinated 14-3-3 eta protein or fragment thereof in thebiological sample from the first subject is compared to the level ofautoantibodies against, or immune complexes with, citrullinated 14-3-3eta in biological samples from one or more other subjects whose subtypeof arthritis is known and/or previously-established, wherein suchcomparison determines the subtype of arthritis for the first subject.

Determination that the levels of autoantibodies against, or immunecomplexes with, at least one citrullinated 14-3-3 protein or fragmentthereof in the biological sample from the first subject are similar tothe levels of autoantibodies against, or immune complexes with, at leastone citrullinated 14-3-3 protein or fragment thereof in the biologicalsample from in the biological sample of another subject whose subtype ofarthritis is known and/or previously-established may indicate that thefirst subject has the same subtype of arthritis as the other subject.For example, similar levels of autoantibodies against, or immunecomplexes with, at least one citrullinated 14-3-3 protein or fragmentthereof in the biological sample from the first subject and in thebiological sample of another subject known to have inflammatoryarthritis, e.g., Rheumatoid arthritis, may determine that the firstsubject also has inflammatory arthritis, e.g., Rheumatoid arthritis.

Additionally, determination that the levels of autoantibodies against,or immune complexes with, at least one citrullinated 14-3-3 protein orfragment thereof in the biological sample from the first subject aredissimilar to the levels of autoantibodies against, or immune complexeswith, at least one citrullinated 14-3-3 protein or fragment thereof inthe biological sample from another subject whose subtype of arthritis isknown and/or previously-established may indicate that the first subjecthas a subtype of arthritis different than that of the other subject. Forexample, dissimilar levels of autoantibodies against, or immunecomplexes with, at least one citrullinated 14-3-3 protein or fragmentthereof in the biological sample from the first subject and in thebiological sample of another subject known to have noninflammatoryarthritis, e.g., osteoarthritis, may determine that the first subjecthas an inflammatory arthritis, e.g., Rheumatoid arthritis.

In one embodiment, the detecting step comprises an immunological-basedtechnique, e.g., immunoprecipitation, ELISA, Western blot analysis,immunohistochemistry, immunofluorescence, “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitation reactions,immunodiffusion assays, in situ immunoassays, precipitation reactions,agglutination assays, complement fixation assays, protein A assays,Immunoelectrophoresis assays, fluorescence activated cell sorting (FACS)analysis, radioimmunoassay, and the like.

Detecting and/or measuring autoantibodies against a citrullinated 14-3-3protein or fragment thereof according to the methods described hereinmay thus be performed by observing the formation of an immune complexbetween the autoantibody and citrullinated 14-3-3 or fragment thereof ina sample, or alternatively determining the presence of an existingautoantibody/citrullinated 14-3-3 complex in a sample. In oneembodiment, the formation may be detected by way of detectably labeledcitrullinated 14-3-3 protein(s) or fragment(s) thereof. In anotherembodiment, the complex may be detected by forming a second immunecomplex between the autoantibody/citrullinated 14-3-3 complex and adetectably labeled secondary antibody that binds immunoglobulin, e.g.,the immunoglobulin backbone of the autoantibody.

In one embodiment, the methods involve detecting autoantibodies againstcitrullinated 14-3-3 or circulating immune complexes thereof in theblood, synovial fluid, plasma, serum, or tissue (e.g. synovial joint,damaged joint tissue, etc.) of a patient. In one embodiment, detectionis done by immunoprecipitation of autoantibodies against citrullinated14-3-3 from blood, synovial fluid, plasma, serum or tissue usingcitrullinated 14-3-3 protein or fragment thereof. In one embodiment,detection involves the use of ELISA. In one embodiment, detectioninvolves Western blot analysis of a sample comprising synovial fluid,plasma, or serum from a patient. In one embodiment, detection involvesthe use of radioimmunoassay. In one embodiment, detection involves theuse of a strip test. In one embodiment, detection involves the use of apoint of care test. In one embodiment, detection of autoantibodiesagainst citrullinated 14-3-3 or circulating complexes thereof iscombined with detection of another marker of arthritis (e.g., MMP,anti-CCP, anti-RF and/or CRP).

Also described herein are kits comprising a reagent for evaluating anarthritic condition in a subject, wherein the reagent specificallyrecognizes autoantibodies to citrullinated 14-3-3 protein or a fragmentthereof. In one embodiment, the reagent may include a detectably labeledcitrullinated 14-3-3 protein or fragment thereof, which may also beimmobilized on a solid support. The citrullinated 14-3-3 protein orfragment thereof may comprise an epitope shared between a plurality of14-3-3 protein isoforms, or may comprise an epitope unique to one or asubset of citrullinated 14-3-3 protein isoforms. In preferredembodiments, the citrullinated 14-3-3 protein or fragment thereofcomprises a citrullinated 14-3-3 eta and/or gamma epitope. In oneembodiment, the citrullinated 14-3-3 protein or fragment thereofcomprises a citrullinated 14-3-3 eta epitope shared by at least oneother citrullinated 14-3-3 isoform, e.g. 14-3-3 gamma. In anotherembodiment, the citrullinated 14-3-3 eta epitope is unique to 14-3-3eta.

In another aspect, provided herein is an antibody capable of bindingselectively to a citrullinated human 14-3-3 protein or a citrullinatedfragment thereof, over the native human 14-3-3 eta protein or nativehuman 14-3-3 eta fragment thereof, respectively. In a preferredembodiment, the isolated antibody competes for binding to acitrullinated 14-3-3 eta protein with anti-14-3-3 eta autoantibodiesspecific for the citrullinated 14-3-3 eta protein but not withanti-14-3-3 eta autoantibodies specific for the native ornon-citrullinated form of the 14-3-3 eta protein.

In one embodiment, the antibody is capable of binding selectively tocitrullinated 14-3-3 eta protein or a citrullinated fragment thereof,over native 14-3-3 eta protein or native 14-3-3 eta fragment thereof,respectively In a preferred embodiment, the antibody is capable ofselectively binding a citrullinated 14-3-3 eta protein comprising anamino acid sequence selected from the group consisting of SEQ IDNOs:16-22 over a native 14-3-3 eta protein comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:9-15,respectively. In one embodiment, an antibody provided herein selectivelybinds a protein comprising the amino acid sequence of SEQ ID NO:16, butnot a protein comprising the amino acid sequence of SEQ ID NO:9. In oneembodiment, an antibody provided herein selectively binds a proteincomprising the amino acid sequence of SEQ ID NO:17, but not a proteincomprising the amino acid sequence of SEQ ID NO:10. In one embodiment,an antibody provided herein selectively binds a protein comprising theamino acid sequence of SEQ ID NO:18, but not a protein comprising theamino acid sequence of SEQ ID NO:11. In one embodiment, an antibodyprovided herein selectively binds a protein comprising the amino acidsequence of SEQ ID NO:19, but not a protein comprising the amino acidsequence of SEQ ID NO:12. In one embodiment, an antibody provided hereinselectively binds a protein comprising the amino acid sequence of SEQ IDNO:20, but not a protein comprising the amino acid sequence of SEQ IDNO:13. In one embodiment, an antibody provided herein selectively bindsa protein comprising the amino acid sequence of SEQ ID NO:21, but not aprotein comprising the amino acid sequence of SEQ ID NO:14. In oneembodiment, an antibody provided herein selectively binds a proteincomprising the amino acid sequence of SEQ ID NO:22, but not a proteincomprising the amino acid sequence of SEQ ID NO:15. In anotherembodiment, the antibody is capable of selectively binding citrullinated14-3-3 gamma protein over native 14-3-3 gamma protein.

In another aspect, the present invention concerns the finding that thedegree of citrullination of individual 14-3-3 eta proteins, and/or theidentification of citrulline resides at particular sites within theprotein, e.g. at any one or more of positions 4, 12, 19, 42, 61, 86 and227 of SEQ ID NO: 5, is also indicative of the existence and/or statusof the arthritic condition in the subject. Also provided herein aremethods for evaluating and/or characterizing an arthritic condition in amammalian subject comprising detecting the degree and/or specificcitrullination positions of at least one citrullinated 14-3-3 etaprotein or a fragment thereof in a biological sample from a subjectemploying the aforementioned selective antibodies.

Accordingly, described herein are methods for evaluating and/orcharacterizing an arthritic condition in a mammalian subject comprisingcontacting a biological sample from the subject with one or moreantibodies capable of binding selectively to a citrullinated human14-3-3 eta protein or a citrullinated fragment thereof and detecting thecitrullinated 14-3-3 eta protein or fragment thereof, wherein thepresence, degree and/or location of citrullination within said 14-3-3eta protein or fragment thereof is indicative of the existence and/orstatus of the arthritic condition in the subject, in comparison with adefined clinical test result standard.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. shows a bar graph representing the 14-3-3 eta citrullinationspecific autoantibody response in anti-CCP negative and anti-CCPpositive rheumatoid arthritis (RA) patients as measured by theautoantibody reactivity directed towards either the recombinantnon-citrullinated or citrullinated form of the 14-3-3 eta antigen.14-3-3 eta autoantibodies preferentially bind to the citrullinated formof the 14-3-3 eta antigen in both anti-CCP negative and positivepatients.

FIG. 2 is a dot plot representing the 14-3-3 eta citrullination specificautoantibody response in thirty anti-CCP negative healthy controls (●)compared to thirty anti-CCP negative rheumatoid arthritis (RA) patients(▴). The y-axis (anti-cit-14-3-3q) represents the autoantibody responsetowards the citrullinaed form of the 14-3-3 eta antigen for eachindividual subject.

DETAILED DESCRIPTION

“14-3-3” and “14-3-3 protein” are used interchangeably and refer to atleast one member of the 14-3-3 family. A 14-3-3 protein is a member of afamily of conserved intracellular regulatory molecules that areubiquitously expressed in eukaryotes. 14-3-3 proteins have the abilityto bind a multitude of functionally diverse signaling proteins,including kinases, phosphatases, and transmembrane receptors. Indeed,more than 100 signaling proteins have been reported as 14-3-3 ligands.14-3-3 proteins may be considered evolved members of the TetratricoPeptide Repeat superfamily. They generally have 9 or 10 alpha helices,and usually form homo- and/or hetero-dimer interactions along theiramino-termini helices. These proteins contain a number of known domains,including regions for divalent cation interaction, phosphorylation andacetylation, and proteolytic cleavage, among others.

There are seven distinct genetically encoded isoforms of the 14-3-3proteins that are known to be expressed in mammals, with each isoformcomprising between 242-255 amino acids. The seven 14-3-3 proteinisoforms are designated as 14-3-3 α/β (alpha/beta), 14-3-3 δ/ξ(delta/zeta), 14-3-3 ε (epsilon), 14-3-3 γ (gamma), 14-3-3 η (eta),14-3-3 τ/θ (tau/theta), and 14-3-3 σ (sigma/stratifin). 14-3-3 proteinshave a high degree of sequence similarity, and are known to undergopost-translational processing, e.g., phosphorylation, citrullination,etc. See, e.g., Megidish et al. (1998) J. Biol. Chem. 273: 21834-45.Consequently, anti-14-3-3 autoantibodies may specifically bind to and/orrecognize more than one 14-3-3 protein isoform, or may specifically bindand/or recognize only one isoform (e.g., 14-3-3 eta).

Citrullination is a form of a post-translational modification (PTM)whereby peptidylarginine deiminases (PAD) catalyze the deimination ofthe amino acid arginine (R) to citrulline (C) resulting in a chemicalchange liberating a nitrogen-based moiety. Accordingly, the terms“citrullinated 14-3-3 protein” and “citrullinated 14-3-3 peptide” areinterchangeable and refer to a protein that has an amino acid sequencethat is identical to the amino acid sequence of a native 14-3-3 protein(e.g., a 14-3-3 protein that has not been modified post-translationally)but for the substitution of at least one arginine residue in the nativesequence with a citrulline residue in the citrullinated sequence.

“Substituted by” or “replaced by” include modified into, e.g., anarginine residue that is substituted or replaced by a citrulline residuecan also mean an arginine residue modified into a citrulline residue,e.g. by incubation with PAD. Citrullination by PAD starts mostly at theNH₂-terminus of the protein, but exceptionally it can start from theCOOH terminus of the protein. In case several arginine residues arereplaced by citrulline residues, this means that for said severalarginie residues each single arginine residue is replaced by one singlecitrulline residue.

Peptidylarginine deiminases (PADs), also referred to as protein-argininedeiminases, are a family of posttranslational modification enzymes thatconvert arginine residues in peptides to citrulline residues in thepresence of calcium ion.

“Citrulline” and “Cit” refers to 2-amino-5-(carbamoylamino)pentanoicacid and is an alfa-amino acid with formula: H₂NC(O)NH(CH₂)₃CH(NH₂)CO₂H.

The terms “peptide” and “protein” are interchangeable as used herein andrefer to a molecule comprising an amino acid sequence of between 2 and200 amino acids, connected by peptide bonds. Peptides may contain any ofthe conventional 20 amino acids or modified versions thereof and anynon-naturally occurring amino-acids incorporated by chemical peptidesynthesis or by chemical or enzymatic modification. Peptides may be usedas antigens and may comprise one or more epitopes.

The term “derivative” “variant” and “fragment” as used herein withreference to a protein refers to molecules which comprises at least theactive portion of said protein, e.g., comprises at least the epitopeand/or the citrulline residue of said protein, either or both of whichare specifically bound by anti-14-3-3 autoantibodies.

The term “epitope” refers to one or several portions (which may define aconformational epitope) of an antigenic protein which is/arespecifically recognized and bound by an antibody or a portion thereof(Fab′, Fab2′, etc.) or a receptor presented at the cell surface of a Bor T cell lymphocyte, and which is able, by said binding, to induce animmune response. Epitopes are chemical features generally present onsurfaces of molecules and accessible to interaction with an antibody.Typical chemical features are amino acids and sugar moieties, havingthree-dimensional structural characteristics as well as chemicalproperties including charge, hydrophilicity, and lipophilicity.Conformational epitopes are distinguished from non-conformationalepitopes by loss of reactivity with an antibody following a change inthe spatial elements of the molecule without any change in theunderlying chemical structure.

An ordinarily skilled artisan will recognize that autoantibodiesrecognize fragments of the antigen. Accordingly, as used herein,“fragment thereof” and “epitope” are used interchangeably and generallyrefer to a determinant of 14-3-3 that is capable of binding to anantibody, e.g., an autoantibody. Accordingly, the term “epitope” whenused in reference to 14-3-3 proteins or specific isomers generallyrefers to a fragment of the protein, including a citrullinated 14-3-3protein, that is capable of binding to an antibody, e.g., anautoantibody.

Described herein are citrullinated 14-3-3 epitopes that comprise atleast one citrulline residue and are recognized by autoantibodies in apatient diagnosed with arthritis, particularly rheumatoid arthritis,methods of using such epitopes to evaluate and/or characterize anarthritic condition in a subject, and kits comprising such epitopes. Anepitope can comprise as few as 3 amino acids in a spatial conformationwhich is unique to the epitope. Generally an epitope consists of atleast 6 such amino acids, and more usually at least 8-10 such aminoacids. Methods for determining the amino acids which make up an epitopeinclude x-ray crystallography, 2-dimensional nuclear magnetic resonance,and epitope mapping.

“Antibody” refers to a composition comprising a protein that bindsspecifically to a corresponding antigen and has a common, generalstructure of immunoglobulins. The term antibody specifically coverspolyclonal antibodies, monoclonal antibodies, dimers, multimers,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments, so long as they exhibit the desired biological activity.Antibodies may be murine, human, humanized, chimeric, or derived fromother species. Typically, an antibody will comprise at least two heavychains and two light chains interconnected by disulfide bonds, whichwhen combined form a binding domain that interacts with an antigen. Eachheavy chain is comprised of a heavy chain variable region (VH) and aheavy chain constant region (CH). The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3, and may be of the mu,delta, gamma, alpha or epsilon isotype. Similarly, the light chain iscomprised of a light chain variable region (VL) and a light chainconstant region (CL). The light chain constant region is comprised ofone domain, CL, which may be of the kappa or lambda isotype. The VH andVL regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (CIq)of the classical complement system. The heavy chain constant regionmediates binding of the immunoglobulin to host tissue or host factors,particularly through cellular receptors such as the Fc receptors (e.g.,FcγRI, FcγRII, FcγRIII, etc.). As used herein, antibody also includes anantigen binding portion of an immunoglobulin that retains the ability tobind antigen. These include, as examples, F(ab), a monovalent fragmentof VL CL and VH CH antibody domains; and F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region. The term antibody also refers to recombinant singlechain Fv fragments (scFv) and bispecific molecules such as, e.g.,diabodies, triabodies, and tetrabodies (see, e.g., U.S. Pat. No.5,844,094).

“Antigen” is to be construed broadly and refers to any molecule,composition, or particle that can bind specifically to an antibody. Anantigen may have one or more epitopes that interact with the antibody,although it does not necessarily induce production of that antibody.

“Autoantibodies” are endogenous antibodies that specifically bind selfantigens, i.e., a normal tissue component. An autoantibody is producedin response to a naturally occurring antigen of the same body thatproduces the autoantibody. Accordingly, the terms “autoantibodiesagainst 14-3-3” and “autoantibodies to 14-3-3” are used interchangeablyand refer to endogenous antibodies produced by a mammalian subject thatspecifically bind a 14-3-3 protein, which may be citrullinated, or afragment thereof from said host.

“Immunological binding” and “formation of an immune complex” are usedinterchangeably and as used in this context, generally refer to thenon-covalent interactions of the type which occur between an antibody,e.g., an autoantibody, and an antigen for which the antibody isspecific. The strength, or affinity of immunological bindinginteractions can be expressed in terms of the dissociation constant(K_(d)) of the interaction, wherein a smaller K_(d) represents a greateraffinity. Immunological binding properties can be quantified usingmethods well known in the art. For example, see Davies et al. (1990)Annual Rev. Biochem. 59:439-473. An antibody, or antigen-bindingfragment thereof, is said to “specifically bind,” “immunologicallybind,” and/or is “immunologically reactive” if it reacts at a detectablelevel (within, for example, an ELISA assay) with ligand, and does notreact detectably with unrelated ligands under similar conditions.

The phrase “specifically (or selectively) binds” to an antibody, whenreferring to a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. The term “directed against”a protein or peptide, when referring to an antibody, refers also to thespecific binding reaction that is determinative of the presence of theprotein by said antibody in a heterogeneous population of proteins andother biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to marker “X” from specific species such as rat, mouse, or humancan be selected to obtain only those polyclonal antibodies that arespecifically immunoreactive with marker “X” and not with other proteins,except for polymorphic variants and alleles of marker “X”. Thisselection may be achieved by subtracting out antibodies that cross-reactwith marker “X” molecules from other species. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select antibodies specifically immunoreactive witha protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual(1988), for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity). Typically a specific orselective reaction will be at least twice background signal or noise andmore typically more than 10 to 100 times background.

“Diagnostic” means identifying the presence or nature of a pathologiccondition in a subject based on the presence or absence of one or moreautoantibodies to the citrullinated 14-3-3 protein(s) disclosed herein.The diagnostic method makes the correlation between the presence ofautoantibodies directed against citrullinated 14-3-3 protein(s) and theoccurrence of a specific disease, e.g., rheumatoid arthritis, or a groupof diseases (e.g. arthritic conditions).

“Prognostic” means determining the potential progression or outcome of apathologic condition and/or disease process in a subject, with orwithout treatment, based on the presence/absence and/or amount of one ormore autoantibodies to the citrullinated 14-3-3 protein(s) disclosedherein. The prognostic method makes the correlation between the presenceand/or amount of autoantibodies directed against citrullinated 14-3-3protein(s) and the progression and/or likely outcome of the arthriticcondition.

“Theranostic” means determining the potential and/or likely reaction toa proposed treatment protocol for a pathologic condition and/or diseaseprocess in a subject, based on the presence/absence and/or amount of oneor more autoantibodies to the citrullinated 14-3-3 protein(s) disclosedherein, and tailoring an appropriate treatment for the subject based onthe results. The theranostic method makes the correlation between thepresence and/or amount of autoantibodies directed against citrullinated14-3-3 protein(s) and the likely efficacy of different treatment optionson the arthritic condition in the subject.

“Subject” and “patient” are used interchangeably and refer to, exceptwhere indicated, mammals such as humans and non-human primates, as wellas rabbits, rats, mice, goats, pigs, and other mammalian species.

“Arthritic condition,” “arthritis,” and “arthralgia” are usedinterchangeably, and generally refer to, except where indicated, aninflammatory disorder of the joints of the body. Pain, swelling,stiffness, and difficulty of movement are frequently associated witharthritic conditions. Arthritis consists of more than 100 differentconditions. These can be anything from relatively mild forms tocrippling systemic forms, see, e.g., www.arthritis.ca/types%20of%/20arthritis/default.asp?s=1. An arthritic condition may resultfrom any of several causes, including infection, trauma, degenerativedisorders, metabolic disorders or disturbances, or other unknownetiologies.

An arthritic condition may be more specifically described according tothe subtype, for example, rheumatoid arthritis, mixed connective tissuedisease (MCTD), crystal induced arthritis, reactive arthritis,spondylarthropathy, osteoarthritis, sarcoidosis, palindromic rheumatism,post traumatic arthritis, malignancy related arthritis, septicarthritis, lyme arthritis, osteoarthritis, bacterial, infectiousarthritis, etc. Arthritis may further accompany other identifieddisorders, including gout, ankylosing spondylitis, systemic lupuserythematosus, inflammatory bowel disease, psoriasis, etc. Well-definedarthritic condition refers to knowledge regarding the type of arthritisand its stage, e.g., onset, remission, relapse etc.

Citrullinated 14-3-3 Proteins

The amino acid sequences of native 14-3-3 proteins are set forth inTable 1. In some embodiments, a 14-3-3 protein is identical to thenative 14-3-3 protein sequences provided in Table 1. The 14-3-3 proteinmay also be substantially homologous to a native 14-3-3 protein sequenceprovided in Table 1 and retain the functional activity of the 14-3-3protein, e.g., specific binding to an anti-14-3-3 autoantibody, yetdiffers in amino acid sequence due to natural allelic variation ormutagenesis.

TABLE 1 14-3-3 proteins 14-3-3 Protein NCBI Accession No. SEQ ID NO:14-3-3 a/β NP_003395.1 1 14-3-3 δ/ξ NP_001129171.1 2 14-3-3 εNP_006752.1 3 14-3-3 γ NP_036611.2 4 14-3-3 η NP_003396.1 5 14-3-3 τ/θNP_006817.1 6 14-3-3 σ NP_006133.1 7

As used herein, the phrase “14-3-3 eta protein” refers to a proteincomprising SEQ ID NO: 5 as well as a protein substantially homologousthereto, e.g., a protein having at least 75%, yet more preferably 80% to90%, still more preferably 90%-95%, again more preferable 95%, and mostpreferably at least 98% amino acid sequence identity with SEQ ID NO:5.The amino acid sequence of SEQ ID NO:5 is provided below.

MGDREQLLQR ARLAEQAERY DDMASAMKAV TELNEPLSNEDRNLLSVAYK NVVGARRSSW RVISSIEQKT MADGNEKKLEKVKAYREKIE KELETVCNDV LSLLDKFLIK NCNDFQYESKVFYLKMKGDY YRYLAEVASG EKKNSVVEAS EAAYKEAFEISKEQMQPTHP IRLGLALNFS VFYYEIQNAP EQACLLAKQAFDDAIAELDT LNEDSYKDST LEVIQLLRDNL TLWTSDQQDE EAGEGN

To determine the percent identity of two amino acid sequences or of twopolynucleotide sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or polynucleotide sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, the length of a reference sequencealigned for comparison purposes is at least 30%, preferably at least40%, more preferably at least 50%, even more preferably at least 60%,and even more preferably at least 70%, 80%, or 90% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or polynucleotide “identity” is equivalent toamino acid or polynucleotide “homology”). The percent identity betweenthe two sequences is a function of the number of identical positionsshared by the sequences, taking into account the number of gaps, and thelength of each gap, which need to be introduced for optimal alignment ofthe two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Inanother embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of E. Meyers andW. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into theALIGN program (version 2.0), using a PAM 120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4.

The protein sequences of the present invention can further be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous topolynucleotide molecules of the invention. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3 to obtainamino acid sequences homologous to 14-3-3 protein molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997)Polynucleotides Res. 25(17):3389-3402. When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used, e.g., at www.ncbi.nim.nih.gov.

Incubation of a 14-3-3 protein, fragment, or fusion thereof with apeptidylarginine deiminase according to well-known methods results in acitrullinated 14-3-3 protein, fragment or fusion thereof. Suchcitrullinated 14-3-3 protein, fragment or fusion thereof can be used asan immunogen, to produce anti-14-3-3 antibodies, purify anti-14-3-3antibodies, and in diagnostic, prognostic and theranostic assays asdescribed herein.

In accordance with the present invention, the human 14-3-3 etacitrullination sites identified in silico, in vitro and using clinicalsamples are provided in Table 2.

TABLE 2 AA AA Position Sequence Arginylated PeptideCitrullinated Peptide   4   1-12 MGD[R]EQLLQRAR MGD[Cit]EQLLQRAR(SEQ ID NO: 9) (SEQ ID NO: 16)  12   4-18 REQLLQRA[R]LAEQAEREQLLQRA[Cit]LAEQAE (SEQ ID NO: 10) (SEQ lD NO: 17)  19  12-26RLAEQAE[R]YDDMASA RLAEQAE[Cit]YDDMASA (SEQ ID NO: 11) (SEQ ID NO: 18) 42  29-45 KAVTELNEPLSNED[R]NLL KAVTELNEPLSNED[Cit]NLL (SEQ ID NO: 12)(SEQ ID NO: 19)  61  50-69 KNVVGARRSSW[R]VISSIEQKKNVVGARRSSW[Cit]VISSIEQK (SEQ ID NO: 13) (SEQ ID NO: 20)  86  77-89KKLEKVKAY[R]EKI KKLEKVKAY[Cit]EKI (SEQ ID NO: 14) (SEQ ID NO: 21) 227217-235 KDSTLIMQLL[R]DNLTLWTS KDSTLIMQLL[Cit]DNLTLWTS (SEQ ID NO: 15)(SEQ ID NO: 22)

Citrullinated proteins of the invention comprise at least citrullineresidue at one of the amino acid positions listed in Table 2. In oneembodiment, a citrullinated 14-3-3 eta fragment according to the presentinvention comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 16-22.

As shown in Table 3, several if not all of the seven citrullinated siteslisted in Table 2 are conserved in other 14-3-3 isoforms as well.Accordingly, to the extent that these citrullination sites are conservedin other 14-3-3 isoforms, these sites may also be used to determine thecitrullination status of these other 14-3-3 isoforms using the methodsand materials disclosed herein.

TABLE 3 Amino Acid Site Alpha/ in Eta Eta Gamma beta Epsilon Sigma ThetaZeta 4 Present Present No Present Present No No 12 Present Present No NoNo No No 19 Present Present Present Present Present Present Present 42Present Present Present Present Present Present Present 61 PresentPresent Present Present Present Present Present 86 Present PresentPresent Present Present Present Present 227 Present Present PresentPresent Present Present Present

Several aspects of the invention pertain to isolated citrullinated14-3-3 proteins, and biologically active portions thereof, e.g.,fragments suitable for use as antigens to anti-citrullinated 14-3-3autoantibodies. Citrullinated 14-3-3 proteins, including fragmentsthereof, may be isolated from natural, recombinant or synthetic 14-3-3protein (including fragments thereof, respectively), through the actionof peptidylarginine deiminase, e.g., by incubating native 14-3-3 proteinor fragments thereof, respectively, with peptidylarginine deiminasesaccording to well-known methods. Alternatively, citrullinated 14-3-3proteins may be isolated by peptide synthesis according to well-knownmethods, e.g., by directly incorporating citrulline residues into thepeptide synthesized.

In one embodiment, 14-3-3 proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, 14-3-3 proteins areproduced by recombinant DNA techniques.

Preferably, a 14-3-3 protein or fragment thereof of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the 14-3-3 protein or fragment thereof are ligatedin an expression vector in-frame in accordance with conventionaltechniques, for example by employing blunt-ended or stagger-endedtermini for ligation, restriction enzyme digestion to provide forappropriate termini, filling-in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andenzymatic ligation. Alternatively, PCR amplification of gene fragmentscan be carried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a c gene sequence (see, e.g.,Current Protocols In Molecular Biology, eds. Ausubel et al. John Wiley &Sons: 1992). Moreover, many expression vectors are commerciallyavailable that already encode a moiety, e.g., a detectable moiety. Apolynucleotide encoding a 14-3-3 protein or fragment thereof can becloned into such an expression vector such that a moiety, e.g., adetectable moiety, is linked in-frame to the 14-3-3 protein or fragment.

A signal sequence can be used to facilitate secretion and isolation ofthe secreted protein or other proteins of interest. Signal sequences aretypically characterized by a core of hydrophobic amino acids which aregenerally cleaved from the mature protein during secretion in one ormore cleavage events. Such signal peptides contain processing sites thatallow cleavage of the signal sequence from the mature proteins as theypass through the secretory pathway. Thus, the invention pertains to thedescribed polypeptides having a signal sequence, as well as topolypeptides from which the signal sequence has been proteolyticallycleaved (i.e., the cleavage products). In one embodiment, apolynucleotide sequence encoding a signal sequence can be operablylinked in an expression vector to a protein of interest, such as aprotein which is ordinarily not secreted or is otherwise difficult toisolate. The signal sequence directs secretion of the protein, such asfrom a eukaryotic host into which the expression vector is transformed,and the signal sequence is subsequently or concurrently cleaved. Theprotein can then be readily purified from the extracellular medium byart recognized methods.

Alternatively, the signal sequence can be linked to the native orcitrullinated 14-3-3 protein, fragment, or fusion thereof, using asequence which facilitates purification, such as with a GST domain.

Antibodies to Citrullinated 14-3-3 Protein and Fragments Thereof

In other embodiments, the invention provides antibodies, i.e., intactantibodies and antigen binding fragments thereof, that specifically bindto a citrullinated 14-3-3 protein or fragment thereof, preferablymammalian (e.g., human) 14-3-3 protein, which may be useful indiagnosing, prognosing, monitoring and/or treating RA.

Antibody molecules to the polypeptides of the present invention, e.g.,citrullinated 14-3-3 protein or citrullinated fragments thereof, may beproduced by methods well known to those skilled in the art. For example,monoclonal antibodies may be produced by generation of hybridomas inaccordance with known methods. Hybridomas formed in this manner are thenscreened using standard methods, such as an enzyme-linked immunosorbentassay (ELISA), to identify one or more hybridomas that produce anantibody that specifically binds with the polypeptides of the presentinvention. For example, a citrullinated 14-3-3 protein or acitrullinated fragment thereof of the invention may also be used toimmunize non-human hosts, e.g., donkey, goat, sheep, guinea pig,hamster, rabbit, rat and mouse, to obtain polyclonal and monoclonalantibodies that react with the citrullinated 14-3-3 protein or acitrullinated fragment thereof but not the native 14-3-3 protein ornative fragment thereof. The peptide immunogens additionally may containa cysteine residue at the carboxyl terminus, and may be conjugated to ahapten such as keyhole limpet hemocyanin (KLH). Additional peptideimmunogens may be generated by replacing tyrosine residues with sulfatedtyrosine residues. Methods for synthesizing such peptides are well knownin the art. A full-length polypeptide of the present invention may beused as the immunogen, or, alternatively, antigenic peptide fragments ofthe polypeptides may be used. An antigenic peptide of a polypeptide ofthe present invention comprises at least 7 continuous amino acidresidues and encompasses an epitope such that an antibody raised againstthe peptide forms a specific immune complex with the polypeptide.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues.

Monoclonal antibodies may be generated by other methods known to thoseskilled in the art of recombinant DNA technology. As an alternative topreparing monoclonal antibody-secreting hybridomas, a monoclonalantibody to a polypeptide of the present invention may be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with a polypeptide related tothe present invention (e.g., citrullinated 14-3-3 protein or acitrullinated fragment thereof) to thereby isolate immunoglobulinlibrary members that bind to the polypeptides related to the presentinvention (e.g., citrullinated 14-3-3 protein or a citrullinatedfragment thereof). Techniques and commercially available kits forgenerating and screening phage display libraries are well known to thoseskilled in the art. Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay libraries can be found in the literature. For example, the“combinatorial antibody display” method is well known and was developedto identify and isolate antibody fragments having a particular antigenspecificity, and can be utilized to produce monoclonal antibodies. Afterimmunizing an animal with an immunogen as described above, the antibodyrepertoire of the resulting B-cell pool is cloned. Methods are generallyknown for obtaining the DNA sequence of the variable regions of adiverse population of immunoglobulin molecules by using a mixture ofoligomer primers and PCR. For instance, mixed oligonucleotide primerscorresponding to the 5′ leader (signal peptide) sequences and/orframework 1 (FR1) sequences, as well as primers to a conserved 3′constant region, can be used for PCR amplification of the heavy andlight chain variable regions from a number of murine antibodies; asimilar strategy has also been used to amplify human heavy and lightchain variable regions from human antibodies.

Polyclonal sera and antibodies may be produced by immunizing a suitablesubject with a polypeptide of the present invention. The antibody titerin the immunized subject may be monitored over time by standardtechniques, such as with ELISA using immobilized protein. If desired,the antibody molecules directed against a polypeptide of the presentinvention may be isolated from the subject or culture media and furtherpurified by well-known techniques, such as protein A chromatography, toobtain an IgG fraction.

Fragments of antibodies to the polypeptides of the present invention maybe produced by cleavage of the antibodies in accordance with methodswell known in the art. For example, immunologically active Fab andF(ab′).sub.2 fragments may be generated by treating the antibodies withan enzyme such as pepsin.

Additionally, chimeric, humanized, and single-chain antibodies to thepolypeptides of the present invention, comprising both human andnonhuman portions, may be produced using standard recombinant DNAtechniques and/or a recombinant combinatorial immunoglobulin library.Humanized antibodies may also be produced using transgenic mice whichare incapable of expressing endogenous immunoglobulin heavy and lightchain genes, but which can express human heavy and light chain genes.For example, human monoclonal antibodies (mAbs) directed against, e.g.,a citrullinated 14-3-3 protein or fragment thereof, may be generatedusing transgenic mice carrying the human immunoglobulin genes ratherthan murine immunoglobulin genes. Splenocytes from these transgenic miceimmunized with the antigen of interest may then be used to producehybridomas that secrete human mAbs with specific affinities for epitopesfrom a human protein.

Chimeric antibodies, including chimeric immunoglobulin chains, may beproduced by recombinant DNA techniques known in the art. For example, agene encoding the Fc constant region of a murine (or other species)monoclonal antibody molecule is digested with restriction enzymes toremove the region encoding the murine Fc, and the equivalent portion ofa gene encoding a human Fc constant region is substituted.

An antibody or an immunoglobulin chain may be humanized by methods knownin the art. Humanized antibodies, including humanized immunoglobulinchains, may be generated by replacing sequences of the Fv variableregion that are not directly involved in antigen binding with equivalentsequences from human Fv variable regions. General methods for generatinghumanized antibodies are provided by Morrison (1985) Science229:1202-07; Oi et al. (1986) BioTechniques 4:214; Queen et al., U.S.Pat. Nos. 5,585,089; 5,693,761; 5,693,762, the contents of all of whichare hereby incorporated by reference. Those methods include isolating,manipulating, and expressing the nucleic acid sequences that encode allor part of immunoglobulin Fv variable regions from at least one of aheavy or light chain. Sources of such nucleic acid sequences are wellknown to those skilled in the art and, for example, may be obtained froma hybridoma producing an antibody against a predetermined target. Therecombinant DNA encoding the humanized antibody, or fragment thereof,then can be cloned into an appropriate expression vector.

Humanized or CDR-grafted antibody molecules or immunoglobulins may beproduced by CDR grafting or CDR substitution, wherein one, two, or allCDRs of an immunoglobulin chain can be replaced. See, e.g., U.S. Pat.No. 5,225,539; Jones et al. (1986) Nature 321:552-25; Verhoeyan et al.(1988) Science 239:1534; Beidler et al. (1988) J. Immunol. 141:4053-60;Winter, U.S. Pat. No. 5,225,539, the contents of all of which are herebyincorporated by reference. Winter describes a CDR-grafting method thatmay be used to prepare the humanized antibodies of the present invention(UK Patent Application GB 2188638A; Winter, U.S. Pat. No. 5,225,539),the contents of which are hereby incorporated by reference. All of theCDRs of a particular human antibody may be replaced with at least aportion of a nonhuman CDR, or only some of the CDRs may be replaced withnonhuman CDRs. It is only necessary to replace the number of CDRsrequired for binding of the humanized antibody to a predeterminedantigen.

Human antibodies may additionally be produced using transgenic nonhumananimals that are modified so as to produce fully human antibodies ratherthan the animal's endogenous antibodies in response to challenge by anantigen. See, e.g., PCT publication WO 94/02602. The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the XENOMOUSE™ as disclosed inPCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells that secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv molecules.

Monoclonal, chimeric and humanized antibodies that have been modifiedby, e.g., deleting, adding, or substituting other portions of theantibody, e.g., the constant region, are also within the scope of theinvention. As nonlimiting examples, an antibody can be modified bydeleting the constant region, by replacing the constant region withanother constant region, e.g., a constant region meant to increasehalf-life, stability, or affinity of the antibody, or a constant regionfrom another species or antibody class, and by modifying one or moreamino acids in the constant region to alter, for example, the number ofglycosylation sites, effector cell function, Fc receptor (FcR) binding,complement fixation, etc.

Methods for altering an antibody constant region are known in the art.Antibodies with altered function, e.g., altered affinity for an effectorligand, such as FcR on a cell, or the C1 component of complement, can beproduced by replacing at least one amino acid residue in the constantportion of the antibody with a different residue (see, e.g., EP 388,151A1, U.S. Pat. Nos. 5,624,821 and 5,648,260, the contents of all of whichare hereby incorporated by reference). Similar types of alterations tothe murine (or other species) immunoglobulin may be applied to reduce oreliminate these functions, and are known in the art.

For example, it is possible to alter the affinity of an Fc region of anantibody (e.g., an IgG, such as a human IgG) for an FcR (e.g., Fc gammaR1), or for C1q binding by replacing the specified residue(s) with aresidue(s) having an appropriate functionality on its side chain, or byintroducing a charged functional group, such as glutamate or aspartate,or an aromatic nonpolar residue such as phenylalanine, tyrosine,tryptophan or alanine (see, e.g., U.S. Pat. No. 5,624,821).

Diagnostic, Prognostic and Therapeutic Methods, and Treatment Monitoring

In one aspect, the invention provides methods for diagnosing diseasesand conditions that involve autoantibodies against citrullinated 14-3-3.In general, the presence or absence of an rheumatoid arthritis, orpatient prognosis, may be determined by (a) contacting a biologicalsample obtained from a mammalian subject with at least one citrullinated14-3-3 protein or fragment thereof; (b) detecting in the sample thelevel of autoantibodies that specifically bind to the citrullinated14-3-3 protein or fragment thereof; and (c) comparing the level in suchantibodies with an appropriate control, e.g., the level of native ornon-citrullinated 14-3-3 protein.

The methods comprise using at least one citrullinated 14-3-3 protein orfragment thereof to detect autoantibodies against the protein. There area variety of assay formats known to those of ordinary skill in the artfor using a protein to detect antibodies in a sample. See, e.g., Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. As nonlimiting examples, detection of autoantibodiesagainst citrullinated 14-3-3 may be performed using well-known methodsor assays, e.g. immunoprecipitation, ELISA, Western blot analysis,immunohistochemistry, immunofluorescence, “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitation reactions,immunodiffusion assays, in situ immunoassays, precipitation reactions,agglutination assays, complement fixation assays, protein A assays,Immunoelectrophoresis assays, fluorescence activated cell sorting (FACS)analysis, radioimmunoassay, a strip test, a point of care test, and thelike. The ordinarily skilled artisan will recognize that these methodsmay also be used to measure the level of autoantibodies against, orimmune complexes with, citrulinated 14-3-3 proteins in the biologicalsample.

In some embodiments, an automated detection assay is utilized. Methodsfor the automation of immunoassays include those described in U.S. Pat.Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which isherein incorporated by reference. In some embodiments, the analysis andpresentation of results is also automated. For example, in someembodiments, software that generates a prognosis based on the presenceor absence of a series of proteins corresponding to arthritic conditionsis utilized, including citrullinated 14-3-3 proteins.

In one embodiment, the assays involve the use of at least onecitrullinated 14-3-3 protein or a fragment thereof immobilized on asolid support to bind to and capture autoantibodies that specificallybind the citrullinated 14-3-3 protein(s) from the remainder of thesample. The bound autoantibodies may then be detected using a detectionreagent that contains a reporter group and specifically binds to theantibody/protein complex. Such detection reagents may comprise, forexample, a binding agent that specifically binds to the autoantibodysuch as an anti-human antibody.

The solid support may be any material known to those of ordinary skillin the art. For example, the solid support may be a test well in amicrotiter plate or a nitrocellulose or other suitable membrane.Alternatively, the support may be a bead or disc, such as glass,fiberglass, latex or a plastic material such as polystyrene orpolyvinylchloride. The support may also be a magnetic particle or afiber optic sensor, such as those disclosed, for example, in U.S. Pat.No. 5,359,681. The citrullinated 14-3-3 protein or fragment thereof maybe immobilized on the solid support using a variety of techniques knownto those of skill in the art, which are amply described in the patentand scientific literature. In the context of the present invention, theterm “immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the antibody and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the antibody, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In one embodiment, a microtitre plate coatedwith streptavidin is used in conjunction with a biotinylatedcitrullinated 14-3-3 protein or fragment thereof.

Covalent attachment of the citrullinated 14-3-3 protein or fragmentthereof to a solid support may generally be achieved by first reactingthe support with a bifunctional reagent that will react with both thesupport and the citrullinated 14-3-3 protein or fragment thereof. Thecaptured autoantibody can then be detected using the non-competitive“sandwich” technique where labeled ligand for the autoantibody isexposed to the washed solid phase. Alternatively, competitive formatsrely on the prior introduction of a labeled antibody to the sample sothat labeled and unlabelled forms compete for binding to the solidphase. Such assay techniques are well known and well described in boththe patent and scientific literature. See, e.g., U.S. Pat. Nos.3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; and 4,098,876. Enzyme-linked immunosorbent assay (ELISA)methods are described in detail in U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,879,262; and 4,034,074. ELISA assays detect very low titersof autoantibodies.

Autoantibodies can also be detected by solid-phase radioimmunoassay(RIA). The solid phase is exposed to the serum sample in the presence ofradio-labeled antibodies that compete for binding to the immobilizedligand. In this assay, the amount of radiolabel bound to the solid phaseis inversely related to the amount of autoantibodies initially presentin the serum sample. After separation of the solid phase,non-specifically bound radiolabel is removed by washing, and the amountof radiolabel bound to the solid phase determined. The amount of boundradiolabel is, in turn, related to the amount of autoantibodiesinitially present in the sample.

In one embodiment, the assay is performed in a flow-through or striptest format, wherein the citrullinated 14-3-3 protein or fragmentthereof is immobilized on a membrane, such as nitrocellulose. In theflow-through test, autoantibodies to citrullinated 14-3-3 proteinswithin the sample bind to the immobilized citrullinated 14-3-3 proteinor fragment thereof as the sample contacts the membrane. A second,labeled binding agent then binds to the immune complex as a solutioncontaining the second binding agent contacts the membrane. The detectionof bound second binding agent may then be performed as described above.In the strip test format, one end of the membrane to which citrullinated14-3-3 protein or fragment thereof is bound is immersed in a solutioncontaining the sample. The sample migrates along the membrane through aregion containing second binding agent, e.g., to the autoantibodies, andto the area of immobilized citrullinated 14-3-3 protein or fragmentthereof. Concentration of second binding agent at the area ofimmobilized citrullinated 14-3-3 protein or fragment thereof indicatesthe presence of an arthritic condition, or patient prognosis, etc.Typically, the concentration of second binding agent at that sitegenerates a pattern, such as a line, that can be read visually. Theabsence of such a pattern indicates a negative result. In general, theamount of binding agent immobilized on the membrane is selected togenerate a visually discernible pattern when the biological samplecontains a level of the autoantibody that would be sufficient togenerate a positive signal in the assay, in the format discussed above.Preferred binding agents for use in such assays are citrullinated 14-3-3proteins and fragments thereof. Such tests can typically be performedwith a very small amount of biological sample and at the point of care,which may also be quantifiable.

In addition to detecting the presence of autoantibodies in a sample,many methods can be used to quantitatively measure the levels of theautoantibodies. In some methods, the antigen reacts with theautoantibody in a liquid phase, and the autoantibodies arequantitatively measured by an immunoprecipitation technique. Forexample, a citrullinated 14-3-3 protein or fragment thereof (i.e., fulllength isomer or antigenic fragments) can be detectably labeled (e.g.,with an isotope or an enzyme). The polypeptides can be labeled duringsynthesis (e.g., by adding 35S-methionine to an in vitro translationsystem or cellular expression system) or after synthesis. The detectableantigen is added directly to a liquid biological sample (e.g., a serum)to form immune complexes. The immune complexes can be precipitated withpolyethylene glycol. The immune complexes can also be isolated with asecondary antibody (e.g., goat anti-human immunoglobulin) or other kindof binding molecules (e.g., protein A or protein G) that is bound to asolid support (e.g., agarose or sepharose beads). The immunoprecipitatesare washed several times after being separated from the liquid sampleand examined for intensity of the detectable label (e.g.,radioactivity). Any autoantibody present in the sample can thus bedetected and quantified. Optionally, an unlabelled polypeptide can alsobe added to compete with the labeled polypeptide for binding toautoantibodies.

The diagnostic methods of the present invention are also directed todetecting in a subject circulating immune complexes formed betweencitrullinated 14-3-3 proteins and an autoantibody. The methods discussedabove can be readily modified for detection of such immune complexes.For example, an immobilized binding molecule (e.g., protein A or proteinG bound to a bead) can be added to a liquid biological sample. Afterseparation from the liquid phase, immune complexes captured by thebinding molecules can be analyzed with SDS-PAGE and probed with variousantibodies against citrullinated 14-3-3 proteins. The captured antigenscan also be subject to direct amino acid sequence analysis. Identity ofthe immune complexes can thus be revealed. A number of assays areroutinely practiced to detect circulating immune complexes in a subject,e.g., as described in Tomimori-Yamashita et al., Lepr Rev, 70(3):261-71,1999 (antibody-based enzyme-linked immunosorbent assay); Krapf et al., JClin Lab Immunol, 21(4):183-7, 1986 (fluorescence linked immunosorbentassay); Kazeem et al., East Afr Med J, 67(6):396-403, 1990 (laserimmunonephelometry); and Rodrick et al., J Clin Lab Immunol, 7(3):193-8,1982 (Protein A-glass fiber filter assay, PA-GFF, and polyethyleneglycol solubilization assay).

To improve clinical sensitivity, multiple markers may be assayed withina given sample. In particular, one or more other markers of arthritis,or prognostic indicators, etc., may be assayed in combination withautoantibodies to citrullinated 14-3-3 protein. These other markers maybe proteins or nucleic acids. In a preferred embodiment, one or more ofthe other markers are MMP proteins or nucleic acids or other factorswhich are commonly used as indicators for arthritis, e.g., anti-CCP,anti-RF, CRP, SAA, IL-6, SIOO, osteopontin, RF, MMP-1, MMP-3, hyaluronicacid, sCD14, angiogenesis markers and products of bone, cartilage orsynovium metabolism (e.g., CTX-I and CTX-II), etc. Methods for isolatingand assaying nucleic acids based on reference sequences are well knownin the art, as are methods for detecting proteins of interest within apatient sample.

An ordinarily skilled artisan will recognize that each of these wellknown assays can be employed to detect circulating immune complexes in abiological sample for the methods of the present invention. Similarly,each of these well known assays can be employed using the antibodies tocitrullinated 14-3-3 protein (and fragments thereof) disclosed in thisinvention to monitor the citrullination status of a 14-3-3 protein in abiological sample, e.g., determine how much how much of the 14-3-3protein is citrullinated and/or which and how many sites of the 14-3-3protein is citrullinated, as part of a clinical testing procedure, e.g.,in diagnostic, prognostic and theranostic assays as described herein. Anordinarily skilled artisan will readily recognize how to adapt each ofthe assay formats, diagnostic, prognostic and theranostic assays, andkits to use the antibodies to citrullinated 14-3-3 protein as describedherein to determine the citrullination status of 14-3-3 protein in abiological sample.

Combination assays may be done concurrently or sequentially. Theselection of markers may be based on routine experiments to determinecombinations that results in optimal sensitivity.

In one embodiment, the invention provides methods for diagnosingarthritic conditions. In general, arthritic conditions may be detectedin a patient based on the presence of autoantibodies to citrullinated14-3-3 in the synovial fluid, synovial joint, blood, plasma, or serum ofa patient. In other words, autoantibodies to citrullinated 14-3-3protein may be used as a marker to indicate arthritic conditions.

In a preferred embodiment, the invention provides methods for diagnosingrheumatoid arthritis. In general, rheumatoid arthritis may be detectedin a patient based on the presence of autoantibodies to citrullinated14-3-3 in the synovial fluid, synovial joint, blood, plasma, or serum ofa patient. In other words, autoantibodies to citrullinated 14-3-3protein may be used as a marker to indicated rheumatoid arthritis. In aparticularly preferred embodiment, the citrullinated 14-3-3 protein is14-3-3 eta.

In addition, the presence of autoantibodies to citrullinated 14-3-3, orthe relative levels of autoantibodies to citrullinated 14-3-3, asdetermined through the use of a citrullinated 14-3-3 protein or fragmentthereof may be a prognostic indicator of early-stage rheumatoidarthritis, before it progresses to a debilitating form. An advantage ofearly prognosis or diagnosis is earlier implementation of a treatmentregimen.

To determine the presence or absence of rheumatoid arthritis in asubject, the level of autoantibodies against, or immune complexes with,citrullinated 14-3-3 in a biological sample from the subject maygenerally be compared to a level of autoantibodies/immune complexescorresponding to a normal control. In one preferred embodiment, thenormal control is established from the average mean level ofautoantibodies against, or immune complexes with, citrullinated 14-3-3in samples from patients without rheumatoid arthritis. In an alternativeembodiment, the normal control value may be determined using a ReceiverOperator Curve, for example see the method of Sackett et al., ClinicalEpidemiology: A Basic Science for Clinical Medicine, Little Brown andCo., 1985, p. 106-7. Briefly, in this embodiment, the control value maybe determined from a plot of pairs of true positive rates (i.e.,sensitivity) and false positive rates (100%-specificity) that correspondto each possible cut-off value for the diagnostic test result. Thecontrol value on the plot that is the closest to the upper left-handcorner (i.e., the value that encloses the largest area) provides themost accurate value, and a sample generating a signal that is higherthan the value determined by this method may be considered positive.Alternatively, the control value may be shifted to the left along theplot, to minimize the false positive rate, or to the right, to minimizethe false negative rate. In general, a sample generating a signal thatis higher than the control value determined by this method is consideredpositive for arthritis.

In one aspect, the invention provides methods for differentiatingbetween subtypes of arthritis. In one embodiment, the methods involvedetermining the level of autoantibodies against, or immune complexeswith, at least one citrullinated 14-3-3 protein or fragment thereof. Ina preferred embodiment, the level of autoantibodies/immune complexes tocitrullinated 14-3-3 in the patient is compared to that of samples fromsubjects whose subtype of arthritis is known and/orpreviously-established.

In one aspect, the invention provides methods for determining theresponse potential of a patient to treatment directed at rheumatoidarthritis. In one embodiment, the methods involve determining the levelof autoantibodies against, or immune complexes with, at least onecitrullinated 14-3-3 protein or fragment thereof in a patient sample. Ina preferred embodiment, the level of autoantibodies to/immune complexeswith citrullinated 14-3-3 in the patient sample is compared to that ofsamples from subjects whose ability to respond to treatment is known. Arelatively high level of autoantibodies to/immune complexes withcitrullinated 14-3-3 in a first patient sample as compared to a samplefrom a non-inflammatory subject and/or a sample from anotherinflammatory patient may indicate the first patient is a preferredcandidate for a well-known treatment, e.g., disease-modifyinganti-rheumatic drug (DMARD) therapy such as anti-TNF, methotrexate,minocycline, hydroxychloroquine, sulphasalazine, azathiprine, anti-IL-1,anti-IL-6r, and the like. Conversely, a relatively low level ofautoantibodies/immune complexes to citrullinated 14-3-3 in a firstpatient sample as compared to a sample from another inflammatory patientmay indicate the first patient is not a preferred candidate for awell-known treatment, especially if the level is closer to that of asample from a non-inflammatory subject.

Treatment regimens for various types of arthritis are known in the art.For example, a patient diagnosed with rheumatoid arthritis may beprescribed non-steroidal anti-inflammatory medications (NSAIDs)initially, to ease the discomfort and reduce the inflammation. Othertreatment regimens may include, for example, steroidal anti-inflammatorymedications (SAIDs e.g. Cortisol, prednisone), cyclooxygenase 2 specificinhibitors (CSIs), glucocorticoids, and/or standard disease-modifyingantirheumatic drugs (DMARDs) such as, e.g., anti-TNF-alpha neutralizingagents, immunosuppressive drugs (e.g., cyclosporine, azathioprine,cyclophosphamide), antibiotics, antimalarials and cytotoxic drugs (e.g.,methotrexate, sulfasalazine, leflunomide). Treatment regimens may alsoadvantageously include those that target citrullinated 14-3-3 proteinsdirectly, see, e.g., PCT/CA2008/002154. Details on dosage or examples ofparticular drugs will be known to those of skill in the art, and may befound in, for example Harrison's Principles of Internal Medicine 15thed. BRAUNWALD et al eds. McGraw-Hill or “The Pharmacological basis oftherapeutics”, 10th edition. 5 HARDMAN H G., LIMBIRD L E. editors.McGraw-Hill, New York, and in “Clinical Oncology”, 3rd edition.Churchill Livingstone/Elsevier Press, 2004. ABELOFF, M D. editor.

In one aspect, the invention provides methods for monitoring treatmentof rheumatoid arthritis. In one embodiment, the methods involvedetermining the level of autoantibodies against, or immune complexeswith, at least one citrullinated 14-3-3 protein or fragment thereof inpatient samples and monitoring the level of autoantibodies to/immunecomplexes with citrullinated 14-3-3 in a patient undergoing treatment.

The presence or relative levels of autoantibodies to/immune complexeswith citrullinated 14-3-3 may correlate with the presence or relativelevels of other proteins known to be associated with arthriticconditions in patients. Nonlimiting examples of proteins well-known tobe associated with an arthritic condition include inflammatorycytokines, such as tumor necrosis factor, and matrix metalloproteinases(MMPs), such as MMP-1 or MMP-3, etc. At least 25 different MMPs havebeen identified. Detection of autoantibodies to citrullinated 14-3-3 incombination with detection of at least one inflammatory cytokine and/orMMP in a patient sample may be used to diagnose arthritis. Additionally,the presence or relative levels of autoantibodies/immune complexes tocitrullinated 14-3-3 in combination with at least one MMP and/or atleast one inflammatory cytokine in a patient sample may be used as aprognostic indicator of early-stage arthritis, before the arthritisprogresses to a debilitating form.

Also described herein are kits for evaluating an arthritic condition,and in particular, rheumatoid arthritis. Such kits typically comprisetwo or more components necessary for performing a diagnostic, prognosticand/or theranostic assay. Components may be compounds, reagents,containers, instructions and/or equipment. For example, one containerwithin a kit may contain one or more citrullinated 14-3-3 protein(s) orfragment(s) thereof. Such kits may also contain a detection reagent asdescribed above that contains a reporter group suitable for direct orindirect detection of antibody binding.

Accordingly, described herein are kits for detecting the presence ofautoantibodies to/immune complexes with citrullinated 14-3-3 andoptionally other markers, e.g., MMPs, in a patient sample, the kit beinguseful for providing a diagnostic or prognostic result suitable fordiagnosing or differentiating various arthritic conditions, and morepreferably, rheumatoid arthritis. Additional indications where thepresence of citrullinated 14-3-3 proteins and/or autoantibodies may beimplicated also include, for example, cardiovascular and/orneurodegenerative disorders. A kit may comprise at least onecitrullinated 14-3-3 protein or fragment thereof, which may optionallybe detectably labeled, e.g., with a radioactive label, a luminescentlabel, a fluorescent label, an enzyme, etc. Methods for detectablylabeling proteins are well-known in the art. Such a kit may furtherinclude detection reagents specific for other markers of arthritis e.g.,anti-CCP, anti-RF, CRP, SAA, IL-6, S100, osteopontin, RF, MMP-I, MMP-3,hyaluronic acid, sCD14, angiogenesis markers and products of bone,cartilage or synovium metabolism (e.g., CTX-I and CTX-II), etc. The kitmay further include secondary reagents necessary for the detection ofautoantibodies to citrullinated 14-3-3 immunologically, such as labeledsecondary antibodies (e.g. anti-human antibodies), chromogenic orfluourogenic reagents, polymerization agents and the like. Instructionsfor using the kit for diagnostic or prognostic purposes, includingappropriate comparison standards for quantifying and/or evaluating thelevel of such autoantibodies in the context of a particular diseasestate, may also be advantageously provided in printed form and/orrecorded on a suitable media.

EXAMPLES

As 14-3-3η is liberated into the synovial space in RA where PAD enzymesare present, we investigated whether 14-3-3η is a citrullination targetthat may be used in the diagnosis of RA, and if so, whethercitrullinated 14-3-3η or fragments could be used to identify anti-CCPnegative RA patients. Identification of citrullination sites on 14-3-3ηinvolved a three (3) stage process 1) in silico prediction, 2) in vitrodetermination and 3) validation or identification with clinicalspecimens.

Example 1: In Silico Identification of Citrullination Sites

Ariginine (R) moieties that represent putative citrullination sites wereidentified based on 1) location of the “R” in relation to the native 3Dprotein configuration; 2) accessibility of “R” to PAD enzyme and 3)sequence flanking “R”. Five (5) putative citrullination sites wereidentified corresponding to “R”: 4, 12, 19, 61 and 227.

Example 2: In Vitro Determination of Citrullination Sites

In vitro citrullination was performed whereby recombinant human 14-3-3eta was co-incubated with either recombinant human PAD2 or PAD4 sincethese two isoforms have been reported to be the two most relevant in RA.

Briefly, PAD2 (MQ16.201) and PAD4 (MQ16.203) were procured fromModiQuest Research. The enzymes were further diluted in 100 μl of PADbuffer (0.1 M Tris HCl, pH 7.4, 10 mM CaCl2 with 5 mM DTT, 1 mM PMSF, 10μg/ml aprotonin, 10 μg/ml leupeptin and 10 μg/ml pepstatin) bringing thestock concentrations of PAD2 to 80 mU/μl and PAD4 to 82.5 mU/μl.

Ten (10) μg of full-length recombinant 14-3-3 eta was incubated witheither PAD2 (8 mU) or PAD4 (8.2 mU). The reaction mixture was adjustedto 100 μl with PAD buffer and incubated at 37° C. for 2 h. Followingincubation, the reaction was terminated through the addition of 25 μl of4× Lammelli buffer. The proteins were resolved by SDS-PAGE and the bandcorresponding to 14-3-3 eta was excised. The excised bands were elutedfrom the gel, trypsinized and then analysed using Fourier Transform MassSpectrometry to identify deiminated sites as described by others. Theresults yielded four putative citrullination sites with PAD2 and 3 withPAD4 for which the results are described in Table 4.

TABLE 4 Citrullination sites for 14-3-3η as determined in vitro SiteSequence No PAD PAD2 PAD4 Position 4 mgdReqllq No Yes Yes Position 19qaeRyddma No Yes Yes Position 42 dRnllsvayk No Yes Yes Position 61sswRvissie No Yes No

Example 3: Detection of Antibodies Directed to Citrullinated 14-3-3 EtaUsing Clinical Samples

In vitro citrullination was performed as described in Example 2, and96-well plates were coated with either the citrullinated ornon-citrullinated form of recombinant 14-3-3 eta. The humanauto-antibody response directed to either the native or citrullinatedforms of 14-3-3 eta in anti-CCP positive and negative patients werequantified using an anti-human antibody.

To evaluate whether these novel autoantibodies are detectable inanti-CCP negative RA patients and differentially expressed compared tohealthy controls, reactivity to both non-citrullinated andcitrullinated-14-3-3 eta was measured in 30 anti-CCP negative RApatients and 30 confirmed anti-CCP negative healthy controls. Mean andmedian autoantibody levels expressed in units (U) was evaluated andcorresponding t-tests and Mann-Whitney U-tests were used to determinedifferences within and between groups. The area under the ROC curve(AUC) was generated for diagnostic utility estimates and to determinelikelihood ratios (LR) for various anti-citrulinated-14-3-3 etacut-offs.

FIG. 1 shows that, compared to non-citrullinated 14-3-3 eta, up to 25×higher reactivity to citrullinated-14-3-3 eta in 2 of the 3 anti-CCPpositive RA patients was observed, revealing for the first time theexpression of autoantibodies to the citrullinated form of 14-3-3 eta inRA. Within the anti-CCP negative RA group, significantly higherreactivity was observed to citrullinated 14-3-3 eta compared to native14-3-3 eta (1943 U versus 395 U, p=0.01). No significant differences inreactivity were observed within the healthy group. FIG. 2 shows thatanti-citrullinated-14-3-3 eta antibody expression was significantlyhigher in anti-CCP negative RA patients with means (SD) and medians(min-max) of 1943 U (3045 U) and 306 U (68-8982 U) compared to 155 U(122 U) and 100 U (45-564 U) for healthy controls, p<0.002. Thecorresponding ROC AUC for anti-citrullinated-14-3-3 eta antibodydifferential expression in anti-CCP negative RA patients compared tohealthy controls was 0.79 (95% CI 0.68-0.91; p<0.0001). At a cut-off of320 U, the specificity and sensitivity were 90% and 50% delivering an LRpositive of 5 increasing to 14 at 439 U with a corresponding specificityof 97% and sensitivity of 47%.

Healthy CCP-ve RA patients N = 58 N = 30 mean (SD) 155U (122U) 1943U(3045U) median (min-max) 100U (45-564U) 306U (68-8982U) AUC 0.79 95% CI0.68-0.91 P-value <0.0001 Cut-off 439U LR 14 Specificity 97% Sensitivity47%

Example 4: Identification of Citrullination Sites Using Clinical Samples

14-3-3 eta is immunoprecipitated from clinical samples positive for the14-3-3 eta protein and the immunoprecipitated protein is resolved bySDS-PAGE and the band corresponding to 14-3-3 eta excised. The excisedbands are eluted from the gel, trypsinized and then analysed usingFourier Transform Mass Spectrometry to identify deiminated sites on theprotein.

Selection of Clinically Relevant 14-3-3 Eta Citrullination Sites

To identify the most relevant 14-3-3 eta citrillunation sites, peptidesbearing either an arginylated or a citrullinated moiety are used toscreen and select the most relevant citrullination sites on 14-3-3 etathat can be used to distinguish both the non-citrullinated form of theprotein as well as differentiate between healthy individuals or thoseaffected with an arthritide.

Comparing 14-3-3 eta expression levels in clinical samples using twodistinct approaches, measurement by MRM/LC-MS and ELISA, illustratesthat differences in expression may be attributable to citrullinationsince deimination of arginine yielding citrulline results in amis-cleavage since trypsin does not cut when the protein iscitrullinated, see Table 5. Specifically in samples 1-6 high levels ofthe 14-3-3 eta protein are detectable by ELISA but appear to havenegligible levels when measured by mass spectrometry compared withsamples 7-12. With mass spectrometry samples are trypsinized and 14-3-3eta levels are quantified through measurement of a peak intensity as aresults of a specific peptide mass, the peptide of which is “AVTELNEPLSNED” which resides next to Arg-42 which has been described hereas a citrullination site. Citrullination specific antibodies to Arg-42will be examined by ELISA in samples 1-12 to verify that Arg-42 is aclinically relevant citrullination site.

TABLE 5 Sample Mass ID Spec ELISA 1 0 297.8 2 0 23.85 3 0 33.11 4 1391078.1 5 0 65.85 6 0 18.7 7 5679 64.47 8 1283 29.07 9 9791 7.9 10 1427847.7 11 1602 3.2 12 2451 31.3

Example 5: Diagnosis, Prognosis, and/or Treatment Monitoring

Detection of Autoantibodies to Citrullinated 14-3-3 Eta Protein in aClinical Sample

The data presented in FIG. 1 demonstrates that detection ofautoantibodies to the citrullinated form of full length 14-3-3 eta isuseful in identifying patients whom are anti-CCP negative thuscomplementing the anti-CCP test in diagnosing seronegative RA patients.The differential expression in RA versus healthy individuals presentedin FIG. 2 demonstrates that anti-citrullinated 14-3-3 eta autoantibodiesare higher in RA patients and will likely be highly specific for RA.Citrullinated 14-3-3 eta fragments, each harboring the differentcitrullinated sites, will be also be used to detect autoantibodies toeach of the different sites. Such detection for site specificautoantibodies is likely be as or more specific for RA than thefull-length citrullinated 14-3-3 eta protein.

Determination of the Citrullination Status of a 14-3-3 Protein in aClinical Sample

If a patient measures positive for autoantibodies to citrullinated14-3-3 eta protein, the protein's citrullination status will beevaluated using monoclonal antibodies raised against either or bothfull-length citrullinated 14-3-3 eta protein or citrullinated fragmentsthereof to evaluate two parameters:

1) How much of the protein is citrullinated?

2) What sites on protein are citrullinated and/or how many sites arecitrullinated?

Titres of the citrullinated 14-3-3 eta auto-antibodies will be examinedon their own and in relation to the 14-3-3 eta serum protein levels aswell as the citrullination status of the protein. Table 5 below definespossible outcomes.

TABLE 5 anti- citrullinated 14-3-3 eta 14-3-3 auto- eta antibodiesprotein levels levels Prognosis High High Bad Low Good Low High Bad LowGood or 14-3-3 eta is not central to disease process

For therapy response and monitoring, high levels of anti-citrullinated14-3-3 eta autoantibodies may implicate use of certain therapies overothers. Additionally, it is expected that a higher % of the proteinbeing citrullinated is correlated with a more significant diseaseburden. It is also expected that different citrullination sites mayimpart different biological activity on the protein and thus associatewith different clinical outcomes. This information may then be used toassists in determining the type of therapy that would be best for aparticular patient and for monitoring therapy response.

For instance, high titres and/or high citrullination status would beuseful for treatment using B-cell inhibitors like rituximab orinhibitors that directly target peptidyl arginine deminiases. Monitoringoutcomes by measuring pre- and post-treatment levels may also be useful.For instance if levels decrease, then patient may be receiving a benefitfrom drug i.e. respond to therapy whereas if the levels remain unchangedor increase, then the therapeutic dose may need to be increase or theclass of therapy may need to be switched.

All patents and patent publications referred to herein are herebyincorporated by reference.

Certain modifications and improvements will occur to those skilled inthe art upon a reading of the foregoing description. It should beunderstood that all such modifications and improvements have beendeleted herein for the sake of conciseness and readability but areproperly within the scope of the following claims.

1.-4. (canceled)
 5. A composition comprising at least one citrullinated14-3-3 eta protein or fragment thereof bound to a solid support, whereinthe protein or fragment thereof is specifically recognized byanti-14-3-3 eta autoantibodies present in the serum of a patientsuffering from an arthritic condition.
 6. The composition of claim 5,wherein the composition comprises a reaction mixture comprising the atleast one citrullinated 14-3-3 eta protein or fragment thereof and abiological sample from a subject suspected of having or at risk fordeveloping an arthritic condition.
 7. A method comprising contacting abiological sample from the subject suspected of having or at risk fordeveloping an arthritic condition with at least one citrullinated 14-3-3protein or fragment thereof under a condition suitable for the formationof at least one immune complex between the citrullinated 14-3-3 proteinor fragment thereof and autoantibodies against the citrullinated 14-3-3protein or fragment thereof that may be present in the biologicalsample; and detecting the presence of immune complexes between thecitrullinated 14-3-3 protein or fragment thereof and autoantibodiesagainst the citrullinated 14-3-3 protein or fragment thereof.
 8. Themethod of claim 7, wherein said detecting step further comprisesmeasuring the amount of autoantibodies against the citrullinated 14-3-3protein or fragment thereof.
 9. The method of claim 7, wherein thecitrullinated 14-3-3 protein is citrullinated 14-3-3 eta.
 10. The methodof claim 7, wherein the citrullinated 14-3-3 protein is a fragment ofcitrullinated 14-3-3 eta.
 11. The method according to claim 7, whereinthe citrullinated 14-3-3 protein or fragment thereof is detectablylabeled with a label selected from the group consisting of a radioactivelabel, a luminescent label, and a fluorescent label, and an enzyme. 12.The method according to claim 7, wherein the citrullinated 14-3-3protein or fragment thereof is bound to a solid support.
 13. The methodaccording to claim 7, wherein the autoantibodies are detected by anELISA assay.
 14. The method according to claim 7, wherein said detectionoccurs by chemiluminesence.
 15. The method according to claim 7, whereinsaid arthritic condition is rheumatoid arthritis.
 16. An antibody thatbinds a citrullinated 14-3-3 protein, wherein the citrullinated 14-3-3protein is specifically recognized by anti-14-3-3 eta autoantibodiespresent in the serum of a patient suffering from an arthritic condition.17. The antibody according to claim 16, wherein said antibodyselectively binds the citrullinated form.
 18. A non-human host producingan antibody according to claim
 16. 19. The host of claim 18, wherein thehost is a hybridoma cell line.
 20. A method for diagnosing and/orprognosing an arthritic condition in a subject comprising contacting abiological sample from the subject with at least one antibody capable ofbinding selectively to a citrullinated human 14-3-3 protein or afragment thereof under a condition suitable for the formation of atleast one immune complex between the antibody and citrullinated 14-3-3proteins that may be present in the biological sample; and detecting thepresence, degree and/or location of at least one citrulline residuewithin said citrullinated 14-3-3 protein, wherein said presence, degreeand/or location of said at least one citrulline residue is indicative ofan arthritic condition in said subject or informative of a prognosis forsaid subject.
 21. The composition of claim 5, wherein the citrullinated14-3-3 protein or fragment thereof: comprises a citrulline residue at aposition selected from the group consisting of position 4, position 12,position 19, position 42, position 61, position 86 and position 227 ofSEQ ID NO: 5; or comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 16-22.